1. Field of the Invention
This invention relates generally to immunoassays, and more specifically to an improved chromatographic assay, often referred to as a lateral flow assay, having a test strip employing susperparamagnetic particles as the labels for the analytes to be detected, where, as an additional feature, the analytical strip is removable for reading the quantity of analytes captured therein and for archival purposes.
2. Discussion of Related Art
Various chromatographic immunoassay techniques have been available for many years. One common aspect of known devices, particularly in the lateral flow technology, is that the assay is read visually, that is, by means of one or more optically readable lines on a test strip, typically held in a carrier, which may have various configurations. One end of the test strip is exposed to the sample, normally a body fluid of some type, being tested for the particular target analytes of interest. It is known that particular analytes are indicative of particular biological, environmental, and biohazard conditions, among others. For example, urine may be tested for pregnancy or ovulation and if the target analytes are present, the test is positive. Body fluids may be tested for the presence of other analytes indicative of biological conditions or they may be indicative of the presence of substances, such as drugs. Another example would be for testing water for contaminates. Examples of lateral flow assay methods and apparatuses, where the reading is normally conducted optically, are shown in U.S. Pat. Nos. 5,591,645; 5,798,273; 5,622,871; 5,602,040; 5,714,389; 5,879,951; 4,632,901; and 5,958,790.
A different technology is employed in other types of biological technologies employing magnetic particles or micobeads, sometimes more specifically termed superparamagnetic iron oxide impregnated polymer beads. These beads are employed to bind with the target analytes in the sample being tested and are then typically isolated or separated out magnetically. Once isolation has occurred, other testing may be conducted, including observing particular images, whether directly optically or by means of a camera. Examples of these technologies are disclosed in U.S. Pat. Nos. 3,981,776; 5,395,498; 5,476,796; 5,817,526; and 5,922,284. Another apparatus for detecting target molecules in a liquid phase is shown in U.S. Pat. No. 5,981,297 where magnetizable particles are employed and the output of magnetic field sensors indicates the presence and concentration of target molecules in the sample being tested. Other examples to sense magnetically using physical forces are disclosed in U.S. Pat. Nos. 5,445,970; 5,981,297 and 5,925,573.
There are several limitations or disadvantages to the known optically detected assays. Because they are optical, only surface changes (coloration, typically) can be detected. The target analytes may be in the sample solution but of such a low concentration that only a relatively few are captured in the capture zone in the porous membrane of the assay. This provides a faint or even non-optically detectable line, and a resultant false negative reading. Quantitative assessments are really only an estimation based on color intensity of the detection line. Because the prior art assays are optically read, they are subject to contamination by exposure, and light-caused degradation. Optical assays have a limited archival shelf life.
None of the known prior art employs magnetic particles in conjunction with lateral flow assay technology.
Broadly speaking, the invention relates to lateral flow immunoassay technology employing superparamagnetic particles as the labels for the analytes to be detected. The bound complexes of labeled particles and analytes are captured in predetermined areas or regions on the test strip and the presence and quantity of labeled analytes are then readable by magnetic means. An advantageous additional feature of the invention is that the test strip can be removable from the support member for archival purposes or for reading by an appropriate magnetic sensing device, or both.
A relatively standard lateral flow assay structure is employed but the invention greatly improves the sensitivity of the device over known lateral flow techniques. It provides a very rapid (a few seconds) measurement of the analytical region in the test strip. There are many advantages of using magnetic particles over known colored particles or other optical indicators in the prior art. These include linearity because magnetic detection is linear with respect to the amount of magnetic material present over a wide range, through at least four orders of magnitude. Time stability is also significant because magnetic particles are stable. The developed assay is available to be archived and retested as necessary. Further, magnetic particles are generally inert to biological systems and the environment so they not only remain stable, they are environmentally and biologically safe. Further, magnetic particles are already in widespread use throughout the diagnostics industry with other technologies so they are readily available. Other benefits of magnetic detection are that since the particles are superparamagnetic, they are magnetic only when in a magnetic field. This allows them to be freely manipulated in solution without aggregating.
Another significant advantage over the prior art optical lateral flow devices is that with this invention the total amount of analytes in the capture region of the test strip is measured as a single mass in one volumetric measurement by magnetic means. The permeability of magnetic fields is such that any analyte contained within the active region of the detector will be measured. This contrasts with optical sensing techniques in which only reporter-analyte interactions on or very near the surface are detectable. In this invention the strength of the magnetic signal increases directly with the mass of iron involved. This inherent linearity of magnetic detection contributes to sensitivity, accuracy and dynamic range. Finally, superparamagnetic particles are physically similar to collodial gold with regard to size, and may be easily adapted to a wide range of lateral flow assays. It is noted that collodial gold, as well as fluorescent latex particles, are typically employed in the prior art optically sensed immunological assay techniques.
In lateral flow technology, at one end of the porous membrane (the active part of the test strip) is the sample introduction area conventionally comprising a sample pad and a conjugate pad. In the prior art, the conjugate pad was the source of freely moveable colored particles, typically gold sols from collodial gold, or fluorescent latex particles. In the present invention, the moveable particles are the superparamagnetic particles which label the target analytes from the sample being introduced through the sample pad. The sample, together with the bound magnetic particle labels and target analytes, move with capillary action along the porous membrane and are captured in a predefined location called a capture region or capture zone. There may be more than one capture zone to enable multiplexing, that is, testing for more than one type of analyte at the same time in the same test strip. Excess analytes and the carrying liquid continue to move on through the capture zone to the other end of the porous membrane, sometimes forming a control line or zone separate from the capture zone. An added feature is that typically a wicking pad is mounted on the far end of the porous membrane to enhance the capillary action which drives the flow from the introduction at one end of the porous membrane through the entire length of the membrane.
The porous membrane typically is mounted on a relatively rigid support or base member, but in an advantageous embodiment a separation sheet, or adhesive layer, exists between the base member and the porous membrane. This enables very easy removal of the test strip, which normally would include the separation sheet, so that the test strip is a very thin element which may be magnetically sensed in an appropriate device. Since optical means are not employed and color at the capture zone has no meaning, the top of the porous member is preferably covered by another protective sheet or membrane which is not transparent. It may be completely opaque. This top sheet may also include pre-printed standards, which are employed for calibrating purposes so that the magnetic detector can be calibrated for each test to ensure complete accuracy. The protective sheet may not be a separate element in some cases, but may only be the upper surface of the membrane properly treated to function as a protective sheet or surface.